| Objective We establish streptozotocin(STZ) induced type 1 diabetes mellitus(DM) rats, obtain transparent specimen of flat-mounted retina to visualize the change of blood-retina barrier(BRB) under ordinary fluorescent microscope. Furthermore, we inject human umbilical cordmesenchymal stem cells(h UCMSCs) induced neural stem cells(NSCs) into the vitreous and observe the protective effect in alteration of BRB in diabetic rats.Methods(1) Fourty male SD rats were randomly divided into control(CON) group(10 rats) and DM group(30 rats). DM rats were induced by STZ injection and furtherly divided into 3 groups based on the time of observation, 1 month(DM-1m) group, 3 months(DM-3m) group and 6 months(DM-6m) group, with 10 rats respectively. CON group rats were injected with the equal volume of solvent. EB of 30 g/L was injected via rat femoral vein in the dose of 45 mg/kg. Fifteen minutes after injection of EB, the rats were sacrificed and the retinas were isolated and cut radially to prepare the flat-mounted retinas in PBS immediately and then were dried till the specimens were clear. The specimens were examined under the fluorescence microscope. For quantitative analysis of vascular leakage, retinal images were batch processed by a customized macro generated in Image-Pro Plus 6.1 for calculation of the percentage of EB leakage.(2) Sixty male SD rats were randomly divided into control group(12 rats), DR group(24 rats) and NSCs group(24 rats). DM rats were induced by STZ injection and control group rats were injected with the equal volume of solvent. There months after DM model foundation, the NSCs group was injected 2μl NSCs(2×104/μl) in the right vitreous, and DR group was injected 2μl PBS. One month later,all the rats were sacrificed. Rats’ retinal vesssels and leakage were visualized by flat-mounted retina. Vescular permeability was quantified by analyzing albumin leakage by EB method. Eyeballs were examined by hematoxylin and eosin(HE) staining.Results(1) Blood glucose of DM rats increased significantly accompanied by weight decreasing(P<0.05). The morphology was normal and EB filled the vessels, exhibiting the red fluorescence under the fluorescence microscope. Compared withCON group, retinal background fluorescence was enhanced slightly in the DM-1m group, and focal leakage of the EB from capillaries, larger vessels and focal dilated vessels were detected in the DM-3m group, further, vascular caliber inequality, retinal hypoperfusion area and a lager number of hyperfluorescence areas were seen in the DM-6m group. The percentage of leakage area was(0.05±0.02) %,(0.27±0.06) %,(1.17±0.18) % and(4.77±0.66) % in CON group, DM-1m group, DM-3m group and DM-6m group, respectively, showing significant difference among the four groups(F=795.800, P<0.000), and the leakage area was obviously bigger in the DM-3m group and DM-6m group than that in the control group(q’=10.338, q’=43.475;both at P<0.000).(2) Retinal blood vessels of control group rats is normal, no EB leakage outside the vessels. The background fluorescence was enhanced and focal leakage and focal dilated vessels were detected in DR group rats. In NSCs group rats, background fluorescence was enhanced slightly and EB leakage area decreased significantly compared with DR group. EB average leakage of NSCs group rats decreased significantly compared with DR group(P<0.05). Retinal tissue pathological HE staining shows that the layers of retina structure are normal and clear in control group; retinal thickness is thin, cell arrangement is disorder and the nucleus is swelling in DR group; NSCs group are between two groups.Conclusions(1) Modified EB-perfused retinal wholemount method is easy and helpful for clear visualization of retinal vessel leakage induced by BRB breakdown in the diabetic rats under the common fluorescence microscope.(2) STZ-induced diabetic rats of three months can be used as early DR modle for experimental sthdy.(3) h UCMSCs-induced NSCs intravitreously could reduce leakage of blood vessels and attenuates BRB breakdown induced by diabetes. |
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